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Spore Dispersal Patterns of Fusarium Circinatum on an Infected Monterey Pine Forest in North-Western Spain

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Spore Dispersal Patterns of Fusarium Circinatum on an Infected Monterey Pine Forest in North-Western Spain Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 October 2017 doi:10.20944/preprints201710.0011.v1 Peer-reviewed version available at Forests 2017, 2017, 432; doi:10.3390/f8110432 Spore dispersal patterns of Fusarium circinatum on an infected Monterey pine forest in north-western Spain Miloň Dvořák1*, Patrik Janoš2, Leticia Botella1, Gabriela Rotková3 and Rafael Zas4 1Phytophthora Research Centre. Department of Forest Protection and Wildlife Management, Faculty of Forestry and Wood Technology, Mendel University in Brno, Zemědělská 1, 613 00 Brno, Czech Republic; email: [email protected], [email protected] 2Department of Forest Protection and Wildlife Management, Faculty of Forestry and Wood Technology, Mendel University in Brno, Zemědělská 1, 613 00 Brno, Czech Republic; email: [email protected] 3Department of Experimental Biology, Masaryk University, Kamenice 735/5, 625 00 Brno, Czech Republic; email: [email protected] 4Department of Forest Genetics and Ecology, Biological Mission of Galicia (MBG-CSIC), Apdo. 28, 36080 Pontevedra, Spain; email: [email protected] *Correspondence: [email protected], tel. +420 545 13 4120 1 © 2017 by the author(s). Distributed under a Creative Commons CC BY license. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 October 2017 doi:10.20944/preprints201710.0011.v1 Peer-reviewed version available at Forests 2017, 2017, 432; doi:10.3390/f8110432 Abstract The airborne inoculum of Fusarium circinatum, the fungal pathogen causing Pine Pitch Canker (PPC), is one of the main means of spread of the disease in forest stands and forest nurseries. Since this world-wide known pathogen was introduced in Europe, its biology in this newly infected area still remains scarcely known. To shed more light on this topic, we set an experiment on a naturally PPC infected forest of Monterey pine in Galicia (NW Spain) with the following two goals: (i) to describe the seasonal spore dispersal pattern during one year of regular sampling and (ii) to assess the spatial spore dispersal pattern around the infested plot. Portable rotating arm spore traps were used and complemented with meteorological measurements. The abundance of F. circinatum spores in the samples was evaluated by quantitative PCR (qPCR) with hydrolysis probe. The results showed almost permanent occurrence of the air inoculum throughout the whole year, being detected in 27 of the 30 samplings. No clear temporal trends were observed, but higher air inoculum was favoured by previous lower air temperatures and lower leaf wetness. Conversely, neither rainfall nor air humidity seemed to have any significant importance. The spatial spread of the inoculum was noted to be successful up to a distance of 1000 m in the wind direction, even with winds of just 5 m s-1. Our study shows that rotating arm spore traps combined with qPCR may be an efficient tool for F. circinatum detection. Key words: Pine pitch canker, Galicia, spore trap, air sampling, qPCR, seasonal dynamics 2 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 October 2017 doi:10.20944/preprints201710.0011.v1 Peer-reviewed version available at Forests 2017, 2017, 432; doi:10.3390/f8110432 Introduction Fusarium circinatum [1] (teleomorph Gibberella circinata) is the causal agent of the disease called Pine Pitch Canker (PPC), which affects up to 60 species of pines and Douglas fir (Pseudotsuga menziesii) [2]. In adult trees, the main symptoms of PPC are pitch soaked cankers on the main stem or big lateral branches [3], which may girdle both stem and branches. Roots, shoots, flowers, cones and seeds may result infected as well. PPC is often responsible of a retarded growth of mature trees and massive mortality of saplings in forest nurseries, causing serious economic losses [4]. Originating naturally in Central America [5], F. circinatum has nowadays a worldwide distribution. Since it was firstly found out in the USA in 1946 [6], it has been later introduced to Japan [7], South Africa [8], South Korea [9], and Chile [10]. In 2005 it was reported in Spain [11], although it was observed in forest nurseries in the Basque country (Spain) in 1997 [12]. After that, F. circinatum rapidly appeared in other European countries, including France [13], Italy [14] and Portugal [15], alarming the European forest authorities. Nowadays, the pathogen is mentioned in the EPPO A2-list as a quarantine organism present in the area of EPPO countries but not widely distributed yet [16]. Although apparently eradicated in Italy and France, it is still present in Portugal and Spain [17]. Transport of infected plant material seems to be the most effective way for PPC introduction, especially for long distances [18]. In forest stands, however, the pathogen can also spread by natural means. F. circinatum is a seedborne pathogen that can survive both superficially and internally in seeds [19], favouring the spread of the disease to the following pine generation. Its macro- and microconidia (asexual spores) are also spread by wind, water and insect vectors that infect trees through weather-related injures and wounds associated with insect feeding and pruning [20–23]. Understanding the temporal and spatial spore dispersal is, thus, critical for fine tuning efficient control measures that limit the disease expansion. The seasonal spore dispersal pattern of F. circinatum has been previously investigated in northern California (USA), where more spores were detected in October than in June and July [18]. This pattern was confirmed by the whole year sampling carried out by Garbelotto et al. [24], who detected the highest spore presence in the same area during the cold and wet weather from November to March. In San Francisco, Garbelotto et al. [24] showed the importance of sea fog, which can alleviate the water deficit during dry periods in summer, and enhance the 3 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 October 2017 doi:10.20944/preprints201710.0011.v1 Peer-reviewed version available at Forests 2017, 2017, 432; doi:10.3390/f8110432 fungal sporulation. Wingfield et al. [25] emphasizes, however, that the life cycle of the pathogen may largely vary among different geographical areas, host species and particular conditions of forest stands. To our best knowledge, studies exploring the temporal dynamics of spore dispersal in Europe are lacking. Spatial spread of F. circinatum spores in forest stands has only been partly investigated. Although it is not known how far the airborne spores may be dispersed [2], it was concluded that the conidia of F. circinatum have limited flight distance potential [2,23,24]. According to Garbelotto et al. [24] its dispersal is little influenced by the wind direction and speed. These authors did not find differences in spore occurrence 100, 200 and 300 m from the infected stand. Anyway, from the epidemiological point of view, long distance transfer of the spores is probably less important due the possibly low viability of the thin walled and hyaline spores [2]. The prior aim of the present study is to contribute to have a better knowledge of the seasonal spore dispersal pattern of F. circinatum, which may help to develop effective control measures of PPC in the European pine forests. Particularly, the objectives of this study were: i) to describe the seasonality of the occurrence of F. circinatum spores during a one-year sampling in an infected locality in Galicia (north western Spain) and ii) to investigate the spatial patterns of spore dispersal and the influence of the wind in the spread of the inoculum. To this end we used an active air-sampling trapping system especially designed for fungal spore assessments, but never used before with F. circinatum. Quantification of the spores collected in these traps was done by qPCR techniques. Two surveys were conducted, one designed for covering the within annual variation in spore abundance, and one designed for analysing the spatial dispersal around the infested spot. 4 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 October 2017 doi:10.20944/preprints201710.0011.v1 Peer-reviewed version available at Forests 2017, 2017, 432; doi:10.3390/f8110432 Material and methods Sampling area The sampling was conducted within a 40 years old forest stand of Pinus radiata with some P. pinaster close to Ponte Caldelas, Galicia, Spain, 440 – 480 m a.s.l (GPS coordinates of the centre: 42.376249º, -8.478177º). The pine stand covers an area of approximately 7.5 ha and it is isolated from other pine forests due to a large forest fire occurred in 2006. Infection of F. circinatum at this stand was confirmed in 2006 by the Regional Forest Service (Xunta de Galicia) and during the progress of the experiment typical symptoms (pitching on the stems) were apparent on many trees. Spore traps Actively rotating arm spore traps ROTTRAP 120 (Miloň Dvořák, Boršov nad Vltavou, Czech Republic) were used for all the experiments. The construction of this spore trap is based on the description of Perkins [26] and McCartney et al. [27]. An electric motor rotates 2400 rpm with a 0.8 mm thick U-shaped, square section wire (fig. 1). The impactors – pair of 50 mm long and 200 mm distanced vertical parts of the wire were covered for every sampling with a new double-sided non-woven tape (Tesa SE, Norderstadt, Germany). Covering the front side (according to the direction of rotation) of each impactor, the spore trap provided an impaction area of 80 mm2. According to equations of Noll [28], the spore traps sample the air with a speed of 120 L·min−1 with almost a 100% collecting efficiency for particles bigger than 7.18 µm. Each trap was mounted 1.4 m high and powered by a 12V/19Ah battery. Figure 1: Running ROTTRAP 120 coated with double-sided tape on the impaction side of the square section wire.
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